Established cell model and mechanism of visceral hypersensitivity and nerve hyperplasia in IBS using P815 and N2a co-culture
10.3969/j.issn.1000-4718.2025.04.025
- VernacularTitle:基于小鼠P815与N2a细胞体外共培养构建模拟肠易激综合征内脏高敏神经增生细胞模型
- Author:
Hongbin LI
1
;
Chunli GAN
;
Xiangyu XIE
;
Shan LIU
;
Qin LU
;
Wei KE
;
Shi-yu QI
;
Yusheng HUANG
;
Hongmei TANG
Author Information
1. 广州中医药大学第一临床医学院,广东 广州 510405;广州中医药大学岭南医学研究中心,广东 广州 510405
- Publication Type:Journal Article
- Keywords:
irritable bowel syndrome;
visceral hypersensitivity;
enteric nervous system;
neural plasticity
- From:
Chinese Journal of Pathophysiology
2025;41(4):825-832
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To establish a cell model of visceral hypersensitivity and nerve hyperplasia in irritable bowel syndrome(IBS)by conducting an in vitro co-culture of mouse P815 mast cells and N2a nerve cells and explore its possible mechanism.METHODS:Enzyme-linked immunosorbent assay(ELISA)with three replicates was used to confirm the C48/80-induced P815 degranulation.The length of neurites was observed under bright field microscopy to determine the number of differentiated neurons,thereby selecting the concentration of retinoic acid(RA)for stimulating the differentia-tion of N2a cells,with four replicates.A co-culture system of P815 and N2a cells was established using Transwell cham-bers with four replicates.The following groups were established:N2a cells cultured alone,N2a cells co-cultured with P815 cells,N2a cells co-cultured with P815 cells plus C48/80,and N2a cells plus RA group.After co-culturing,the num-ber of differentiated N2a cells was observed under bright field.The expression of nerve growth factor(NGF),tyrosine ki-nase receptor A(TRKA),growth-associated protein-43(GAP43),neuron-specific enolase(NSE),synapsin(SYN),and postsynaptic density protein-95(PSD-95)at both protein and gene levels in N2a cells was detected using Western blot and polymerase chain reaction(PCR),with four replicates.RESULTS:The best condition for N2a differentiation was stimulation with 10 μmol/L RA for 24 hours,whereas the best condition for degranulation was stimulation of P815 cells with 20 mg/L C48/80 for 24 hours.Compared with N2a cells cultured alone,the differentiation ratio of the N2a+P815+C48/80 and N2a+RA groups was significantly increased(P<0.01),and the protein and mRNA expressions of NGF,TRKA,GAP43,NSE,SYN,and PSD-95 were significantly increased(P<0.05).CONCLUSION:Our results revealed that mast cell degranulation enhances the level of nerve hyperplasia in enteric nerve cells and promotes changes in nerve structure and function.Synaptic remodeling regulated by abnormal expression of key proteins such as NGF,TRKA,and GAP43 is involved in the nerve hyperplasia induced by mast cell degranulation.
